U.S. patent number 5,847,400 [Application Number 08/791,684] was granted by the patent office on 1998-12-08 for fluorescence imaging system having reduced background fluorescence.
This patent grant is currently assigned to Molecular Dynamics, Inc.. Invention is credited to Christopher C. Alexay, Robert C. Kain.
United States Patent |
5,847,400 |
Kain , et al. |
December 8, 1998 |
Fluorescence imaging system having reduced background
fluorescence
Abstract
A coaxial illumination and collection laser scanning system
designed to provide increased sensitivity by reducing
auto-fluorescence while having a substantially uniform detection
sensitivity across the field of view of an objective lens by
reducing lateral chromatic aberrations at the expense of amplifying
axial chromatic aberrations. Axial chromatic aberrations in the
system are removed in the path of a retro-beam. A laser is in
optical communication with the objective lens. The laser produces a
collimated beam of coherent light that is directed by a scanner
through the objective lens to illuminate a raster of spots on the
sample's surface, thereby stimulating a series of small regions of
the sample to emit light. The system may be used as a confocal or
non-confocal imaging system.
Inventors: |
Kain; Robert C. (San Jose,
CA), Alexay; Christopher C. (Walpole, NH) |
Assignee: |
Molecular Dynamics, Inc.
(Sunnyvale, CA)
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Family
ID: |
27082245 |
Appl.
No.: |
08/791,684 |
Filed: |
January 30, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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595355 |
Feb 1, 1996 |
5646411 |
Jul 8, 1997 |
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Current U.S.
Class: |
250/458.1;
250/461.1; 250/461.2 |
Current CPC
Class: |
G02B
21/002 (20130101); G01N 21/6428 (20130101); G02B
21/0076 (20130101); G01N 21/6458 (20130101); G01N
2021/6463 (20130101); G01N 2021/6421 (20130101) |
Current International
Class: |
G02B
21/00 (20060101); G01N 021/64 () |
Field of
Search: |
;250/461.2,461.1,458.1
;356/445 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Shoemaker et al., "An Ultrafast Laser Scanner Microscope for
Digital Image Analysis", IEEE Transactions on Biomedical
Engineering, vol. BME-29, No. 2, pp. 82-91 (Feb. 1982)..
|
Primary Examiner: Hannaher; Constantine
Attorney, Agent or Firm: Schneck; Thomas McGuire, Jr.; John
P.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This is a continuation-in-part of patent application Ser. No.
08/595,355 filed on Feb. 1, 1996, which issued as U.S. Pat. No.
5,646,411 on Jul. 8, 1997.
Claims
We claim:
1. A fluorescence microscopy system for stimulating a sample to
fluoresce, said system comprising:
a photodetector;
a source for emitting an excitation beam of light along an optical
path, said excitation beam having optical properties to cause said
sample to fluoresce;
an air-spaced objective disposed in said optical path to receive
said excitation beam therethrough to illuminate a region of said
sample and collect fluorescent light emitted from said region,
forming a retro-beam, said objective including a plurality of lens
elements, with each of said plurality of lens elements being
spaced-apart from an adjacent lens element of said air-spaced
objective, thereby avoiding the presence of adhesives in the
portion of the optical path so as to reduce auto-fluorescence
produced by said air-spaced objective;
means, positioned in said optical path between said source and said
objective, for separating said excitation beam from said
retro-beam, wherein said objective directs said retro-beam onto
said separating means, with said separating means directing said
retro-beam toward said photodetector, said photodetector producing
signals representing light impinging thereon; and
beam shaping optics disposed in said optical path, said beam
shaping optics having optical properties to produce
auto-fluorescence in response to said excitation beam impinging
thereon, and filtering means, disposed in said optical path, for
reducing the auto-fluorescence sensed by said detector.
2. A fluorescence microscopy system for stimulating a sample to
fluoresce, said system comprising:
a photodetector;
a source for emitting an excitation beam of light along an optical
path, said excitation beam having optical properties to cause said
sample to fluoresce;
an air-spaced objective disposed in said optical path to receive
said excitation beam therethrough to illuminate a region of said
sample and collect fluorescent light emitted from said region,
forming a retro-beam, said objective including a plurality of lens
elements, with each of said plurality of lens elements being
spaced-apart from an adjacent lens element of said air-spaced
objective, thereby avoiding the presence of adhesives in the
portion of the optical path so as to reduce auto-fluorescence
produced by said air-spaced objective;
means, positioned in said optical path between said source and said
objective, for separating said excitation beam from said
retro-beam, wherein said objective directs said retro-beam onto
said separating means, with said separating means directing said
retro-beam toward said photodetector, said photodetector producing
signals representing light impinging thereon; and
beam shaping optics disposed in said optical path, said beam
shaping optics having optical properties to produce
auto-fluorescence that radiates spherically in response to said
excitation beam impinging thereon and a filtering means, disposed
in said optical path, for attenuating auto-fluorescence propagating
over a solid angle of sufficient size to prevent said
auto-fluorescence from impinging upon said photodetector.
3. A fluorescence microscopy system for stimulating a sample to
fluoresce, said system comprising:
a photodetector;
a source for emitting an excitation beam of light along an optical
path, said excitation beam having optical properties to cause said
sample to fluoresce;
an air-spaced objective disposed in said optical path to receive
said excitation beam therethrough to illuminate a region of said
sample and collect fluorescent light emitted from said region,
forming a retro-beam, said objective including a plurality of lens
elements, with each of said plurality of lens elements being
spaced-apart from an adjacent lens element of said air-spaced
objective, thereby avoiding the presence of adhesives in the
portion of the optical path so as to reduce auto-fluorescence
produced by said air-spaced objective;
means, positioned in said optical path between said source and said
objective, for separating said excitation beam from said
retro-beam, wherein said objective directs said retro-beam onto
said separating means, with said separating means directing said
retro-beam toward said photodetector, said photodetector producing
signals representing light impinging thereon; and
beam shaping optics disposed in said optical path, said beam
shaping optics having optical properties to produce
auto-fluorescence in response to said excitation beam impinging
thereon and a filtering means, disposed to in said optical path,
for attenuating light associated with said excitation beam, thereby
preventing the same from impinging upon said beam shaping
optics.
4. A fluorescence microscopy system for stimulating a sample to
fluoresce, said system comprising:
a photodetector;
a source for emitting an excitation beam of light alone an optical
path, said excitation beam having optical properties to cause said
sample to fluoresce;
an air-spaced objective disposed in said optical path to receive
said excitation beam therethrough to illuminate a region of said
sample and collect fluorescent light emitted from said region,
forming a retro-beam, said objective including a plurality of lens
elements, with each of said plurality of lens elements being
spaced-apart from an adjacent lens element of said air-spaced
objective, thereby avoiding the presence of adhesives in the
portion of the optical path so as to reduce auto-fluorescence
produced by said air-spaced objective;
means, positioned in said optical path between said source and said
objective, for separating said excitation beam from said
retro-beam, wherein said objective directs said retro-beam onto
said separating means, with said separating means directing said
retro-beam toward said photodetector, said photodetector producing
signals representing light impinging thereon; and
wherein said objective has optical properties to provide a
substantially uniform detection sensitivity across said field of
view by introducing, into said system, a specified amount of axial
chromatic aberrations while reducing lateral chromatic aberrations
and a focusing lens means, disposed in the path of said retro-beam,
for directing said retro-beam onto said photodetector and further
including a focusing lens means, disposed between said objective
and said photodetector, for reducing axial chromatic aberrations
introduced by said objective.
5. A fluorescence microscopy system for stimulating a sample to
fluoresce, said system comprising:
a photodetector;
a source for emitting an excitation beam of light along an optical
path, said excitation beam having optical properties to cause said
sample to fluoresce;
an air-spaced objective disposed in said optical path to receive
said excitation beam therethrough to illuminate a region of said
sample and collect fluorescent light emitted from said region,
forming a retro-beam, said objective including a plurality of lens
elements, with each of said plurality of lens elements being
spaced-apart from an adjacent lens element of said air-spaced
objective, thereby avoiding the presence of adhesives in the
portion of the optical path so as to reduce auto-fluorescence
produced by said air-spaced objective;
means, positioned in said optical path between said source and said
objective, for separating said excitation beam from said
retro-beam, wherein said objective directs said retro-beam onto
said separating means, with said separating means directing said
retro-beam toward said photodetector, said photodetector producing
signals representing light impinging thereon; and
wherein said separating means includes a mirror having a diameter
greater than the diameter of said incident beam and smaller than
the diameter of said retro-beam, with the diameter of the
retro-beam being substantially larger than the diameter of the
incident beam.
6. A fluorescence microscopy system for stimulating a sample to
fluoresce, said system comprising:
a photodetector;
a source for emitting an excitation beam of light along an optical
path, said excitation beam having optical properties to cause said
sample to fluoresce;
an objective disposed in said optical path to receive said
excitation beam therethrough to illuminate a region of said sample
and collect fluorescent light emitted from said region, forming a
retro-beam;
a spatial filter, positioned proximate to said photodetector,
having a substantially transmissive aperture, restricting light
scattered rearwardly to increase signal response;
a focusing lens means, disposed in the path of said retro-beam, for
directing said retro-beam onto said aperture, and onto said
photodetector, with said photodetector producing signals
representing light impinging thereon, said focusing lens means
having optical properties to produce auto-fluorescence in response
to said excitation beam passing therethrough; and
first filtering means, disposed in said optical path between said
focusing lens means and said separating means, for reducing the
auto-fluorescence sensed by said detector.
7. The systems as recited in claim 6 wherein said objective
includes a plurality of lens elements, a subset of which has an
adhesive attached thereto to fix a spatial position, along said
optical path, between said subset and the remaining lens elements
of said plurality of lens elements, with all said adhesive present
in said objective lens disposed upon said subset outside of said
optical path, with said excitation beam passing through said
objective lens without impinging upon said adhesive, thereby
reducing auto-fluorescence.
8. The system as recited in claim 6 further including means,
positioned in said optical path between said beam source and said
objective, for separating said incident beam from said retro-beam,
and a beam expander disposed in said optical path between said
source and said separating means, said beam expander having optical
properties to produce auto-fluorescence that radiates spherically
in response to said excitation beam impinging thereon, with a
second filtering means, disposed between said beam expander and
said source, for attenuating auto-fluorescence propagating over a
solid angle of sufficient size to prevent the same from passing
through said transmissive aperture.
9. The system as recited in claim 6 further wherein objective has
optical properties to provide a substantially uniform detection
sensitivity across said field of view by introducing, into said
system, a specified amount of axial chromatic aberrations while
reducing lateral chromatic aberrations and a focusing lens means,
disposed in the path of said retro-beam, for directing said
retro-beam onto said photodetector said focusing lens means having
optical properties to compensate for axial chromatic aberrations
introduced by said objective.
10. The system as recited in claim 6 further including means,
positioned in said optical path between said beam source and said
objective, for separating said incident beam from said retro-beam,
wherein said separating means includes a mirror having a diameter
greater than the diameter of said incident beam and smaller than
the diameter of said retro-beam, with the diameter of the
retro-beam being substantially larger than the diameter of the
incident beam.
11. The system as recited in claim 6 further including means,
positioned in said optical path between said beam source and said
objective, for separating said incident beam from said retro-beam,
and a pinhole disposed in said optical path and a collimating lens
positioned to collimate light passing through said pinhole, with
said pinhole disposed between said beam source and said separating
means, and said collimating lens disposed between said separating
means and said pinhole, wherein said beam source is a non-coherent
source of light optically focused on said pinhole.
12. A fluorescence microscopy system for stimulating a sample to
fluoresce, said system comprising:
a photodetector;
a source for emitting an excitation beam of light along an optical
path, said excitation beam having optical properties to cause said
sample to fluoresce;
an air-spaced objective disposed in said optical path to receive
said excitation beam therethrough to illuminate a region of said
sample and collect fluorescent light emitted from said region,
forming a retro-beam; said objective including a plurality of lens
elements, with each of said plurality of lens elements being
spaced-apart from an adjacent lens element of said objective;
a spatial filter, positioned proximate to said photodetector,
having a substantially transmissive aperture, restricting light
scattered rearwardly to increase signal response;
a focusing lens means, disposed in the path of said retro-beam, for
directing said retro-beam onto said aperture, and onto said
photodetector, with said photodetector producing signals
representing light impinging thereon, said focusing lens means
having optical properties to produce auto-fluorescence in response
to said excitation beam passing therethrough;
means, positioned in said optical path between said beam source and
said objective, for separating said incident beam from said
retro-beam, said separating means directing said retro-beam toward
said photodetector, with said photodetector producing signals
representing light impinging thereon; and
first filtering means, disposed in said optical path between said
focusing lens means and said separating means, for reducing the
auto-fluorescence sensed by said detector.
13. The system as recited in claim 12 further including means,
positioned in said optical path between said beam source and said
objective, for separating said incident beam from said retro-beam,
and a beam expander disposed in said optical path between said
source and said separating means, said beam expander having optical
properties to produce auto-fluorescence that radiates spherically
in response to said excitation beam impinging thereon, with a
second filtering means, disposed between said beam expander and
said source, for attenuating auto-fluorescence propagating over a
solid angle of sufficient size to prevent the same from passing
through said transmissive aperture.
14. The system as recited in claim 12 further wherein objective has
optical properties to provide a substantially uniform detection
sensitivity across said field of view by introducing, into said
system, a specified amount of axial chromatic aberrations while
reducing lateral chromatic aberrations and a focusing lens means,
disposed in the path of said retro-beam, for directing said
retro-beam onto said photodetector said focusing lens means having
optical properties to compensate for axial chromatic aberrations
introduced by said objective.
15. The system as recited in claim 12 further including means,
positioned in said optical path between said beam source and said
objective, for separating said incident beam from said retro-beam,
wherein said separating means includes a mirror having a diameter
greater than the diameter of said incident beam and smaller than
the diameter of said retro-beam, with the diameter of the
retro-beam being substantially larger than the diameter of the
incident beam.
16. The system as recited in claim 12 further including means,
positioned in said-optical path between said beam source and said
objective, for separating said incident beam from said retro-beam,
and a pinhole disposed in said optical path and a collimating lens
positioned to collimate light passing through said pinhole, with
said pinhole disposed between said beam source and said separating
means, and said collimating lens disposed between said separating
means and said pinhole, wherein said beam source is a non-coherent
source of light optically focused on said pinhole.
Description
TECHNICAL FIELD
The present invention relates to laser scanning imaging systems,
particularly for use in fluorescence imaging.
BACKGROUND ART
Fluorescence microscopy is often used in the fields of molecular
biology, biochemistry and other life sciences. One such use is in
identifying a specific antigen using antibodies. Antibodies are
proteins produced by vertebrates as a defense against infection.
They are made of millions of different forms, each having a
different binding site and specifically recognizing the antigen
that induces its production. To identify an antigen, a sample of
cells is provided that contains specific antibodies coupled to a
fluorescent dye. The cells are then assessed for their
fluorescence. Taking advantage of the precise antigen specificity
of antibodies, the cells having fluorescent properties are known to
contain a specific antigen.
Originally, the fluorescence of cells was assessed manually by
visual inspection, using conventional microscopy. This proved
time-consuming and costly. The need for high-speed automated
systems became manifest. Many high-speed imaging systems, such as
confocal microscopes, are available for assaying cell samples. The
illumination and collection optics, along with their relative
geometry, determine in large part the parameters of the other
system elements.
To increase the sensitivity of the fluorescent microscopy systems,
a number of steps have been taken, including improved optical
elements that demonstrate reduced chromatic aberrations.
Additionally, steps have been taken to improve the signal-to-noise
ratio by using techniques that increase the saliency of
fluorescence emitted by a sample among background fluorescence,
fluorescence not emitted by the sample. Similar to chromatic
aberrations, a substantial amount of the background fluorescence in
a system arises from the optical components that make upon the
same.
There are many optical components available. For example, U.S. Pat.
No. 5,404,247 to Cobb et al. discloses an air-spaced, diffraction
limited, seven element telecentric f-.theta. lens. However, the
design of a classical f-.theta. lens is primarily for monochromatic
illumination which can often times exacerbate chromatic aberrations
of a fluorescent microscopy system.
A prior art high-speed imaging system is shown in FIG. 1 and
includes an f-.theta. objective 10 positioned above a sample 11 so
that the surfaces of the objective are perpendicular to the
sample's normal. A laser light source 12 produces a beam 13. The
objective 10 directs the beam 13 to illuminate a spot on the
sample's surface. An oscillating reflective surface 14 is disposed
at the pupil 15 of the system, between the light source 12 and the
objective 10, to deflect the beam 13 back and forth along one axis.
The sample is placed on a table to move the sample in a direction
perpendicular to the first scan direction, thereby resulting in a
two dimensional scan pattern on the sample's surface. The objective
is not designed for coaxial collection resulting in light reflected
from the sample surface being collected by a condenser assembly 16
that is separate and apart from the objective. Such a geometry
results in increased system footprint, increased optical complexity
and a limitation of solid angle collection. The collected light is
then imaged on a photo-detector 17.
A prior art high-speed imaging system, similar to that described
with respect to FIG. 1, is disclosed by Richard L. Shoemaker et
al., in "An Ultrafast Laser Scanner Microscope for Digital Imaging
Analysis", IEEE Transactions on Biomedical Engineering, Vol.
BME-29, No. 2, Feb. 1982, pp. 82-91. The principal difference
between these two systems concerns the scanning device. Instead of
a galvanometric scanner, Shoemaker et al. require the use of a
rotating polygon mirror to scan the spot over the sample's
surface.
Another prior art high-speed imaging system is disclosed in U.S.
Pat. No. 4,284,897 by Sawamura et al., in which laser light is
reflected through two galvanometric mirrors and one dichroic mirror
to direct a beam through an objective and illuminate a spot on a
sample's surface. The galvanometric mirrors are swung in
appropriate directions to allow the spot to scan over the entire
surface of the sample. In response to the illuminating spot, the
sample emits fluorescence light. The objective, serving as a
condenser lens, transmits the light back through a first dichroic
mirror. Positioned behind the first dichroic mirror is a second
dichroic mirror that splits the fluorescent light into a light
produced by a first probe at a first wavelength and light produced
by a second probe at a second wavelength. The first and second
wavelengths are transmitted to respective photo-detectors.
U.S. Pat. No. 5,296,700 to Kumagai discloses a fluorescent confocal
microscope which includes, in pertinent part, an intermediary
optical system disposed between a pair of scan mirrors and an
objective optical system. The intermediary optical system is
designed to cancel chromatic aberrations of magnification
introduced by the objective optical system.
U.S. Pat. No. 5,260,578 to Bliton et al. discloses a scanning
confocal microscope which includes, in pertinent part, two beam
sources. One beam source produces ultra violet light. One beam
source produces visible light. An optical assembly is included in
the common optical train to correct chromatically induced scanning
errors.
U.S. Pat. No. 5,381,224 by Dixon et al. discloses a scanning laser
imaging system which allows simultaneous confocal and non-confocal
imaging of reflected light. The system includes, in pertinent part,
a laser producing a beam which traverses a beam expander and
impinges upon a single mirror disposed in an optical axis, which is
defined by an objective lens. The objective lens directs the beam
onto a sample, which is disposed upon a moveable stage. Disposed
between the objective and the sample is a beam splitter designed to
collect light emitted from the sample. The beam splitter directs a
portion of light emitted from the sample onto a condenser lens,
which in turn directs it onto a non-confocal detector. A portion of
the light collected by the beam splitter is directed along the same
path as the beam, but in an opposite direction, forming a
retro-beam. The retro-beam impinges upon a second beam splitter,
positioned between the scan mirror and the laser. The second beam
splitter directs the light onto a focusing lens. The focusing lens
is positioned proximate to a confocal field stop, having an
aperture.
In U.S. Pat. No. 5,095,213 to Strongin, a novel microscope slide is
employed to reduce background fluorescence. The slide is made of a
plastic that has optical properties rendering it opaque and
substantially non-fluorescent. These optical properties are
achieved by providing the plastic with a sufficient quantity of
black carbon powder.
U.S. Pat. No. 5,091,653 to Creager et al. discloses an apparatus
and method for reducing background fluorescence. The apparatus is a
fiber optic dosimeter. The method of reducing background
fluorescence includes modulating an infra-red stimulating source.
This allows for measurement of background fluorescence during
radiation exposure when infra-red stimulating radiation is not
applied. The background fluorescence is then subtracted from the
gross signal under infra-red stimulation.
U.S. Pat. No. 4,877,965 to Dandliker et al. discloses a fluorometer
for measuring a particular fluorescence emanating from a specimen.
The stimulating radiation is generated in bursts which are then
directed toward the specimen to produce a fluorescence. The timing
of the detection of the fluorescence is controlled to take
advantage of differences in optical decay between background
fluorescence and fluorescence emitted from the specimen. To that
end, the stimulating radiation is directed onto the specimen in
bursts, and the light path to the detector is periodically
blocked.
Although the prior art systems are suitable for fluorescent
microscopy, they require additional optics to correct optical
aberrations over a scan field and to efficiently collect light
emitted from a sample. They also necessitate specialized components
to reduce background fluorescence. This results in a net increase
in the system's cost and size.
What is needed, therefore, is to provide a high-speed, low cost,
laser scanning system with improved signal-to-noise ratio that will
provide point by point image of a sample on both a micro and macro
scale.
A further need exists to provide an imaging system of a
substantially smaller size than the prior art systems that affords
a larger scan field than existing coaxial illumination and
collection systems.
SUMMARY OF THE INVENTION
Provided is a coaxial illumination and collection laser scanning
system designed to provide increased sensitivity by reducing
auto-fluorescence while having a substantially uniform detection
sensitivity across a planar field of view by reducing chromatic
aberrations. For purposes of this application, auto-fluorescence is
defined as fluorescent radiation not associated with fluorescent
radiation of the sample region under test. Specifically, the
objective lens is formed so as to avoid the application of excess
auto-fluorescing materials, such as adhesive, in the optical path
of the lens. Also, the objective lens is designed to reduce lateral
chromatic aberrations at the expense of increasing axial chromatic
aberrations. The axial chromatic aberrations are removed from the
system using another lens already existing in the system.
Auto-fluorescence from the remaining optical components of the
system is removed by selective placement of filters along the
optical path. Specifically, it was found that auto-fluorescence
associated with these optical components is seen by a detector of
the system as arriving over two different trajectories. One of the
trajectories is associated with the through-focus-curve of the
objective lens. That is, auto-fluorescence occurring proximate to
the sample plane is directed onto a detector via the objective lens
directing the same thereon. Auto-fluorescence occurring distal from
the sample plane follows a scattering trajectory. This
auto-fluorescence radiates spherically as though emanating from a
point source.
A laser is in optical communication with the objective lens. The
laser produces a collimated beam of coherent light that is directed
through the objective lens to illuminate a spot on a sample,
thereby stimulating a small region of the sample to emit light. The
objective lens also serves as a condenser and collects the light
emitted by the sample. The objective lens is designed with
correction for lateral chromatic aberrations and without correction
for axial chromatic aberrations. The objective lens directs the
collected light back along the identical path traveled by the
incident beam, but in an opposite direction. A wavelength
discriminating dichroic filter is placed along the optical axis
between the laser and the objective lens to separate the emitted
light from the incident beam, and a focusing lens directs the
collected light onto a photo-detector. The photo-detector produces
a signal in response to the emitted light sensed, representing the
sample emitting the light. The focusing lens is a doublet lens that
uses two elements in order to keep a constant focal length with
different wavelengths of light. The doublet lens is disposed in the
path of a retro-beam and is optically designed to introduce axial
chromatic aberrations necessary to correct for axial chromatic
aberrations introduced elsewhere in the system, e.g., by the
objective lens. To scan over the entire sample, a two dimensional
scanning device having a reflecting element is disposed in the path
of the incident beam. A display device is provided and synchronized
with the scanning device to reproduce an image of the sample.
In another embodiment, a plurality of lasers are provided, each of
which emits one or more wavelengths different from the remaining
lasers. Each of the plurality of lasers are in optical
communication with a common beam expander providing the incident
beam with a plurality of wavelengths of light. The common beam
expander has optical properties that introduce axial chromatic
aberrations to cancel axial chromatic aberrations introduced by the
objective lens, with respect to the incident beam.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a simplified side view of a laser scanning microscope of
the prior art.
FIG. 2 is a side view of optical components of the present
invention.
FIG. 3 is a detailed view of a scanning beam passing through the
objective shown in FIG. 2.
FIG. 4 is a graph showing an amount of optical energy impinging
upon a detector disposed in an image plane using a common
microscope system.
FIG. 5 is a schematic view showing the effects of lateral chromatic
aberrations.
FIG. 6 is a graph showing an amount of optical energy impinging
upon a detector in an image plane employing a large field objective
lens of the present invention.
FIG. 7 is a schematic view of optical elements which comprise a
large field objective lens of the present invention.
FIG. 8 is a plan view of optical components showing the effects of
axial chromatic aberrations.
FIG. 9 is a plan view of the housing in which the optical elements
shown in FIG. 7 are placed.
FIG. 10 is a simplified plan view of a lens element that may be
employed in the objective lens of the present invention.
FIG. 11 is a detailed view of a portion of the optical components
shown in FIG. 2 and including a line filter to block
auto-fluorescence.
FIG. 12 is a simplified side view of the optical components shown
on FIG. 2, including a video display system to reproduce an image
of the sample in accordance with the present invention.
FIG. 13 is a side view of the invention shown in FIG. 2 in accord
with an alternate embodiment.
FIG. 14 is a side view of an alternate embodiment of the present
invention.
BEST MODE FOR CARRYING OUT THE INVENTION
FIG. 2 shows a light source 18 producing an excitation/incident
beam 19 of light. Beam 19 is directed through an excitation filter
20 to reduce unwanted wavelengths in the incident beam 19. Upon
exiting the excitation filter 20, beam 19 impinges upon a beam
expander 21 and then a beam splitter 23. Beam splitter 23 directs
beam 19 onto a two dimensional scanning device 25. Two dimensional
scanning device 25 directs beam 19 through an objective lens 27.
Objective lens 27 directs beam 19 to illuminate a spot (not shown)
on a sample 29, thereby stimulating a small region of sample 29 to
emit light. Typically, the light emitted by sample 29 is
fluorescent. Objective lens 27, acting as a condenser, collects the
fluorescent light, forms a retro-beam 31 and directs retro-beam 31
along an identical path of incident beam 19, but in an opposite
direction. Retro-beam 31 passes through a pupil stop 32, consisting
of a spatial filter with an aperture. After exiting pupil stop 32,
retro-beam 31 impinges upon beam splitter 23. Beam splitter 23
separates fluorescent light from the laser light and directs
retro-beam 31 onto a focusing lens 33 via band pass filter 34.
Focusing lens 33 directs retrobeam 31 onto a transmissive aperture
35 of a spatial filter 37, thereby causing retro-beam 31 to impinge
upon a photodetector 39.
It is preferred that light source 18 is a laser producing a
collimated beam of coherent light. It is possible, however, to use
a non-coherent light source optically coupled to collimating optics
to create an incident light beam, e.g., a light emitting diode. If
a non-coherent light source, such as an LED, is employed, a pinhole
and a collimating lens are disposed in front of the LED to create a
collimated excitation/incident beam capable of being focused to a
small spot. Band pass filter 34 typically rejects the excitation
wavelengths while transmitting longer wavelengths.
Beam splitter 23 may be any known in the art, so long as it is
capable of separating the light of the incident beam from the light
of the retro-beam. For example, beam splitter 23 may be a dichroic
filter or a 50% beam splitter. Alternatively, a polarization
sensitive beam splitter may be used to achieve separation of beams
19 and 31. This embodiment could include a 1/4 waveplate positioned
between the beam splitter and the objective. This would cause beam
19 exiting the 1/4 waveplate to be circularly polarized. Also, the
separating means may be a fresnel reflector. Sample 29 is
illuminated point by point, scanning the spot in a raster scan
fashion over the entire area of sample 29.
Any scanning mechanism that provides a two dimensional scan may be
used, e.g., a rotating polygonal mirror, rotating holographic
scanner, or oscillating prisms. Also, an acousto-optic deflector or
a penta-prism scanning deflector may be employed. The preferred
embodiment, however, is to employ a scanning system having one beam
reflecting element 43 in the path of the incident beam which is
pivotable about two perpendicular axes. Reflecting element 43 is a
planar mirror, but this is not essential. The mirror may be concave
or convex. Refractive or diffractive deflecting elements may also
be used as reflecting element 43. Mirror 43 is pivotable about axis
A. Mirror 43 may be moved by any means known in the art, such as
motor 45, but is typically a galvanometer mirror. Mirror 43 and
motor 45 rest atop a moveable platform 47 that is rotated by a
stepper motor 49. Stepper motor 49 moves platform 47 to pivot
mirror 43 about axis B, which is orthogonal to axis A.
Referring to FIG. 3, objective lens 27 typically forms an external
pupil of the system and affords coaxial illumination and
collection. To maximize collection efficiency, it is preferred that
objective lens 27 have a large numerical aperture. With respect to
incident beam 19, objective lens 27 is afocal in the image plane.
Objective lens 27 is typically telecentric, or near telecentric.
The telecentricity of objective lens 27 results in sample 29's
surface always lying at a right angle with respect to the chief ray
of incident beam 19, exiting objective lens 27. With respect to
incident beam 19, the objective plane is proximate to sample 29.
Beam 19 is shown entering objective lens 27 at three different
positions, with beam 19 having a different angle of incidence at
each position. Regardless of beam 19's angle of incidence on
objective lens 27, the chief ray of beam 19 exiting objective lens
27 is orthogonal to sample 29's surface. One advantage of having
this telecentric objective is that it renders the system
magnification relatively insensitive to errors in focus position.
In addition, objective lens 27 must be designed to operate over a
broad band of wavelengths of light, e.g., the primary wavelength
plus approximately 200 nm, or greater. This allows objective lens
27 to operate with lasers of various wavelengths and to collect
light from a wide variety of fluorochromes.
An important aspect of the system is to reduce an amount of optical
loss in the field of view due to chromatic aberrations, which
reduces the detection sensitivity of the system. FIG. 4 shows the
relationship between field position and the relative detection
sensitivity of a system not being corrected for lateral chromatic
aberrations, as a function of the fraction of optical energy sensed
versus size of the spot impinging upon sample 29. The amount of
light detected on the optical axis 51, as defined by an objective
lens, is substantially greater than the amount of light detected at
a half field position 53. The lowest amount of light detected was
at a full field 55 position, with the field of view defined by the
objective lens. Lateral chromatic aberrations increase at larger
field angles. The non-uniformity of light detected across the field
of view of the lens in the system is typically a result of lateral
chromatic aberrations and other field degradations such as coma.
Referring to FIGS. 5 and 2, lateral chromatic aberrations may cause
a reduction in detection sensitivity by allowing, e.g., green
wavelengths 57 of light to pass through aperture 35 while causing
longer yellow wavelengths 58 to be blocked by field stop 37.
To avoid the problems associated with lateral chromatic
aberrations, it is preferred that objective lens 27 correct for all
lateral chromatic aberrations in the scanning system. This may be
accomplished by reducing the field of view of the objective.
However, there are advantages in providing objective lens 27 with a
large field of view. For example, a large (macro) field of view is
useful for scanning large arrays of samples, e.g., planar field
arrays containing up to a million specimens. Nonetheless, the
increased field of view exacerbates the problems with lateral
chromatic aberrations, because there is increasing difficulty in
correcting lateral chromatic aberrations as the lens' field of view
increases. The macro field of view makes lateral chromatic
aberrations more pronounced, thereby making it more difficult to
provide a uniform resolution across the field.
Considering the aforementioned concepts, the parameters for two
implementations of objective lens 27 are as follows:
TABLE 1 ______________________________________ Micro Macro
Objective Objective ______________________________________ Scan
Area (diagonal) 1 mm 1 cm Resolution 0.6 .mu.m 10 .mu.m Numerical
Aperture 0.50 0.25 Intensity Uniformity 95% 95% Spatial Uniformity
98% 98% Polychromatic Range 500-750 nm 500-750 nm Thru focus
sensitivity 1% (signal 1% (signal change over change over 20 .mu.m)
100 .mu.m) Field Flatness Variation +/-10 .mu.m +/-20 .mu.m Working
Distance 3 mm 3.5 mm Focal Length 22 mm 25 mm Pupil Size 25 mm 13
mm ______________________________________
As can be seen above, the micro and macro objectives described may
allow the system to provide between 0.6 to 10 .mu.m resolution.
FIG. 6 shows a reduction in lateral chromatic aberrations of the
macro objective lens which is achieved at the expense of amplifying
axial chromatic aberrations. The increased axial chromatic
aberrations are shown by the amount of light detected, at the 3
.mu.m position, being substantially less than that shown in FIG. 4.
Specifically, sensed by detector 39 is approximately 40% of the on
axis light 65 impinging upon sample 29, 3 .mu.m from the chief ray.
In FIG. 4, the amount of on axis light 51 detected 3 .mu.m from the
chief ray was nearly 80%. Nonetheless, the overall effect of
lateral chromatic aberrations is shown to be substantially reduced.
This is demonstrated by the amount of light detected for any given
spot size being substantially the same for light at the on axis 65,
half field light 67 and full field 69 positions, i.e., there exists
substantial uniformity of detection sensitivity across the field of
view of the macro objective lens. Additionally, FIG. 6 shows that
at 10 .mu.m from the chief ray, collection from all field points is
greater than 90%. FIG. 7 shows the optical elements of the macro
objective lens, and the specifications are as follows:
TABLE 2 ______________________________________ MACRO OBJECTIVE LENS
Radius Thickness Aperture Surface (mm) (mm) Material (mm)
______________________________________ STO 27.67 2" 29.95 3.89
Schott.sub.-- 24 SK14 3 224.24 0.20 air 24 4" 19.85 5.21 SK14 24 5
136.67 0.65 air 20 6" 134.23 2.10 SF11 22 7 24.80 3.84 air 18 8"
-28.59 6.44 SF5 18 9 -60.20 4.86 air 22 10" 27.86 5.73 SK14 18.6 11
-36.27 0.24 air 18.6 12" 11.40 5.02 SK14 14 13 88.81 0.69 air 11.6
14" -33.76 1.32 SF11 11.6 15 11.90 3.69 air 9
______________________________________
The aforementioned lens parameters and specifications are merely
exemplary. The lens design may be adjusted to provide larger and
smaller fields, as desired. This may be achieved by modifying lens
radii, thickness, glass type, etc. Lenses with vastly different
parameters may be designed to afford optimum performance at other
resolutions and field sizes. Additionally, lenses could be designed
for the same resolution and field size, as the aforementioned
lenses, while satisfying different parameters, e.g., working
distance and field flatness.
Referring to FIG. 8, similar to lateral chromatic aberrations,
axial chromatic aberrations reduce the detection sensitivity of the
system by having the focal length of a lens being wavelength
dependent. For example, green wavelengths 71 of light focus before
impinging upon detector 39, while the longer yellow wavelengths 73
of light are sensed by detector 39. Unlike lateral chromatic
aberrations, however, axial chromatic aberrations do not change
with the field position. Thus, the axial chromatic aberrations
introduced by objective lens 27 may be kept constant, while
correcting for lateral chromatic aberrations. Referring again to
FIG. 1, because incident beam 19 is monochromatic, there is no need
to correct axial aberrations in the incident path. Rather, axial
chromatic aberrations may be corrected in the return (retro) path.
It is preferred that focusing lens 33 corrects, or removes, all
axial chromatic aberrations in the system. This allows
manufacturing a less expensive objective lens 27, because
correcting for both axial and lateral chromatic aberrations in a
single lens greatly increases costs. In addition, the size/foot
print of the system is kept to a minimum. As focusing lens 33 is
necessary to condense retro-beam 31 onto aperture 35, no additional
optics are included to reduce axial aberrations. Typically,
focusing lens 33 is a doublet having the optical properties
necessary to correct axial chromatic aberrations of the system.
Focusing lens 33 could be composed of binary elements or any other
design known in the art which focuses a beam and corrects axial
chromatic aberrations.
Referring again to FIG. 2, an advantage with the objective lens
design described above in Table 2 is that the auto-fluorescence of
the system is greatly reduced. Specifically, it was found that
auto-fluorescence associated with the optical components in the
system is seen by detector 39 as arriving over two different
trajectories. Auto-fluorescence emanating proximate to sample 29
generally follows the path of retro-beam. This holds true for
auto-fluorescence that emanates from the optical elements of
objective 27, or any other auto-fluorescence that falls within the
through-focus-curve of objective 27. As such, this
auto-fluorescence is referred to as through-focus auto-fluorescence
(TFA). Auto-fluorescence that occurs distal from sample 29 does not
fall within the through-focus-curve of objective 27. This
auto-fluorescence does not reach detector 39 by following the path
of retro-beam 31. Rather, auto-fluorescence that emanates distally
from sample 29 appears to detector 39 to radiate spherically from a
point source, referred to as spherical auto-fluorescence (SAF).
Typically, the net flux of auto-fluorescent photons that reach
detector 39 resulting from TFA exceeds the number of
auto-fluorescent photons reaching detector 39 resulting from SAF.
Upon analyzing the source of TFA, it was discovered that the same
was caused by sample components, e.g., the slide and sample, as
well as objective lens 27. With respect to objective lens 27, the
source of the TFA was attributed to the material from which the
optical elements were made, as well as the adhesive which was used
to hold the same together.
To determine the amount of auto-fluorescence attributed to
adhesives, a series of tests were conducted on glass lens elements
typically employed to make complex lens systems, such as objective
lens 27. The adhesives tested were manufactured by Summers Optical,
a Division of EMS Acquisition which is located in Fort Washington,
Pa. under tradenames J-91 and UV-74. The test was conducted by
having two different wavelengths .lambda. of light impinging upon
the lens. For each wavelength .lambda., the number of photons
attributable to TFA was measured using a photomultiplier tube. For
.lambda.=532 nm, the results were as follows:
______________________________________ Average number of Adhesive
applied photons sensed ______________________________________ J-91
9200 UV-74 47069 none 3982 For .lambda. = 633 nm, the results were
as follows: J-91 1400 UV-74 1853 none 1330
______________________________________
Tables 3 and 4 show that an appreciable amount of TFA is
attributable to the adhesives disposed on portions of the lens
elements of the objective 27 that lie in an optical path. Further,
it is shown that the amount of TFA produced by a given adhesive is
wavelength dependent. Therefore, it was discovered that by removing
adhesive from these portions of the lens elements that comprise
objective lens 27, TFA associated therewith may be greatly reduced
and rendered wavelength independent. Thus, an objective lens may be
provided that affords improved signal-to-noise ratio and is
suitable for use with a plurality of wavelengths of light.
Referring to FIG. 9, shown is one embodiment of a lens housing 27a,
which is required to fix the spatial position of the lens elements
along the optical path "P". As can be seen, lens housing 27a
includes a plurality of step portions which form annular shoulders,
each of which supports a lens element. Specifically, lens elements
2", 4", 6", 8", 10", 12" and 14" rest against annular shoulders
27b, 27c, 27d, 27e, 27f, 27g, and 27h, respectively. To fix the
position of each of the lens elements, one end of housing 27a
includes a plurality of threads 28. A hollow cylinder 30 is adapted
to engage threads 28 and press against lens element 14". In this
fashion, the lens elements of objective lens 27 are held in place
without necessitating the use of adhesives in the optical path,
defining an air-space objective lens. In air-spaced objective
lenses, each of the plurality of lens elements, 27b, 27c, 27d, 27e,
27f, 27g and 27h, is spaced apart from an adjacent lens element.
This avoids having adhesives disposed in optical path "P". When
mounted in the system, lens element 27b is positioned proximate to
sample 29.
Alternatively, lens elements 27b, 27c, 27d, 27e, 27f, 27g, and 27h
may be adhered to housing 27a, without disposing any adhesive in
optical path "P". To that end, adhesive could be placed at the
periphery of each of the lens elements so that the side edge of the
lens elements are adhered to the side of housing 27a. This is
shown, for example, with adhesive 32 disposed between housing 27a
and lens element 8". Should it be necessary to apply adhesive
between two lens elements to fix the same together, the adhesive
may be disposed as an annular ring 36, shown in FIG. 10. This would
leave the central portion of lens 38 without adhesive so the same
could be placed in optical path "P". Employing an annular ring 36
of adhesive allows taking advantage of paraxial image while
avoiding TFA.
Referring to FIG. 11, to further reduce the auto-fluorescence in
the system due to spherical auto-fluorescence (SAF), filters were
placed at various points in the optical path. Specifically, as
discussed above, it was discovered that SAF appears, to detector
39, to radiate spherically from a point source. Because field-stop
37 is positioned proximate to photodetector 39 in the retro-path,
the SAF that may impinge upon photodetector 39 depends upon two
variables. The aforementioned variables are the distance "d"
between the point source of SAF and the angle a between the point
source and the retro-path 40, with retro-path 40 being defined as
the path light travels from beam splitter 23 and photodetector
39.
For example, it was discovered that SAF emanated from optical
elements of beam expander 21. Were SAF to be produced at point B,
the same would radiate outwardly shown as rays C. As can be seen,
the angle .alpha. of ray C.sub.1 is sufficient to allow the same to
propagate through the aperture 35 in field stop 37. This results in
ray C.sub.1 reflecting from various optical elements in the optical
path so it appears collimated to the photodetector 39, i.e.,
travels parallel to retro-path 40. As shown, ray C.sub.1 has
reflected from objective 27 to impinge upon focusing lens 33, where
it is directed onto photodetector 39. A substantial number of the
rays C are blocked by the encasement 21a which houses beam expander
21. To block ray C.sub.1 and any other ray that may impinge upon
photodetector 39, a line filter 22 may be placed between beam
expander 21 and beam splitter 23 that allows only the passage of
beam 19 therethrough. The line filter 22 must have sufficient
dimensions to block light radiating from point source B over a
solid angle so as to prevent SAF from passing through aperture 35.
In this fashion, SAF from the optical components of beam expander
21 would not impinge upon photodetector 39.
It was discovered that condenser lens 33 also produces SAF. This
resulted from reflection of beam 19 from sample 29 and the
inefficiency of beam splitter 23. Although beam splitter 23
functions to separate beam 19 from beam 31, analysis showed that a
portion of beam 19 that reflects from sample 29 passes through beam
splitter 23. As a result of that portion of beam 19 impinging upon
condenser lens 33, SAF is produced. Although the SAF produced at
condenser lens 33 is de minimus compared to that produced at point
B, the net flux that travels through aperture 25 is higher. This
results from condenser lens 33 laying in retro-path 40, which
increases the probability that photons associated with SAF will
pass through aperture 25. Specifically, a substantial amount of
rays of SAF produced by condenser lens 33 travel parallel to
retro-path 40. This coupled with the relatively short distance
between condenser lens 33 and photodetector 39 results in a greater
amount of SAF reaching the sample when compared to the SAF produced
at beam expander 21. To avoid SAF being produced by the condenser
lens 33, band pass filter 34 is disposed between the beam splitter
23 and the condenser lens 33 to reject excitation wavelengths while
being transmissive to wavelengths associated with beam 31.
Referring to FIG. 12, the operation of the system is discussed.
Preferably, the system is to take advantage of detection using the
conjugate focal (confocal) technique. In this manner, retro-beam 31
is shown emanating from a point 75 which corresponds to a point
source of light illuminated by an incident beam which was focused
to a diffraction limited spot on sample 29. Retro-beam 31 is imaged
on detector 39, after passing through aperture 35. Aperture 35, in
spatial filter 37, isolates the detection of the system to that
substantially coincident with the illuminating spot so that
aperture 35 and point 75 are optically conjugated with each other.
Although any light detector may be used, it is preferred to use a
photomultiplier tube. The signal from the photomultiplier tube
passes through electrical connections 77 to a signal processor 79
of a video display system including a video display screen 81. The
signal from the photomultiplier tube 39 modulates the intensity of
the image signal transmitted from the processor 79 through the
output line 83 to the display screen 81. A scanning signal
generator 85, under control of the signal processor 79 via line 89,
supplies electrical signals to the scanning apparatus 25 through
electrical connections 87. The scanning apparatus 25 moves in
response to the generator 85's signals. The signal from
photomultiplier tube 39 is digitized and stored in memory and can
be simultaneously scanned onto a display.
Although fluorescent confocal imaging is the preferred embodiment,
the system may be used in a non-confocal manner. In this fashion,
field stop 37 and aperture 35 may filter light in a either
non-confocal or semi-confocal manner. In either manner, spatial
filter 37 and aperture 35 improve the signal to noise ratio. Pupil
stop 32 is configured to control the numerical aperture of the
objective lens with respect to retro-beam 31. Without pupil stop
32, the numerical aperture at a given scan angle would be
established by vignetting of objective lens 27. In effect, pupil
stop 32 increases retro beam 31's intensity uniformity across
objective lens 27's field of view and defines both the diameter of
retro-beam 31 impinging upon focusing lens 33, and, therefore, the
numerical aperture of the system. Although pupil stop 32 is shown
positioned between scanning device 25 and beam splitter 23, pupil
stop 32 may be positioned anywhere in the retro path between
scanning device 25 and focusing lens 33.
An obvious extension of the system is in the area of reflection
imaging. That is, the reflected laser beam could be collected at
the detector instead of the fluorescent beam. Both the reflected
beam and the fluorescent beam could be read at a different detector
if a second dichroic beam splitter was positioned after the primary
dichroic beam splitter. Or in a like manner, multiple fluorescent
labels could be detected by using multiple secondary beam splitters
and detectors.
Referring to FIG. 13, a second embodiment of the present invention
is shown in which a further benefit of having all axial chromatic
aberrations corrected in the retro-path is described. The second
embodiment of the system includes all of the features of the
systems described above with respect to FIG. 1, except two or more
light sources 117 and 118 are provided, each of which emits a
wavelength different from the remaining light source, forming an
incident beam 119 comprising multi-chromatic light. The light
sources may be utilized simultaneously, or each could be scanned
individually by turning-off, or shuttering, the undesired light
source. A beam expander 121 is optically coupled to at least one of
the light sources 117 and 118 to control the collimated beam
diameter and to correct for any axial chromatic aberrations in the
system along the incident beam 119 path. In this fashion,
substantially all light comprising incident beam 119 will impinge
upon the same focal plane of sample 129. This is particularly
useful for providing a highly efficient confocal imaging
system.
Each light source 117 and 118 may be uniquely associated with a
beam expander. It is preferred, however, that all light sources are
in optical communication with a common beam expander, as shown, in
order to reduce both the size and cost of the system. To that end,
a dichroic filter 120 is disposed between light sources 117 and 118
and beam expander 121. Dichroic filter 120 allows light from source
117 to pass through while reflecting light from source 118 so that
the light from both form incident beam 119 before entering beam
expander 121. Beam expander 121 has optical properties that
introduce axial chromatic aberrations to cancel axial chromatic
aberrations introduced by optical elements, e.g., objective lens
127 in the system following beam expander 121 to ensure all
wavelengths of light comprising beam 119 impinge upon the same
focal plane of sample 129.
Referring to FIG. 14, shown is another objective lens system that
may be employed by having all axial chromatic aberrations corrected
in the retro-path. The collection lens 233 may be used in a
microscope style system. In such a system, objective lens 227 is
optically coupled to reflecting element 243 via an eye piece 230.
The remaining elements of the system are identical to those shown
in FIG. 2.
* * * * *